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Science , eaaf Xie, Z. Wu, Y. Bug mapping and fitness testing of chemically synthesized chromosome X. Shen, Y. Deep functional analysis of synII, a kilobase synthetic yeast chromosome. Richardson, S. Design of a synthetic yeast genome. Mitchell, L. Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond.
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Mercy, G. Dymond, J. Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Annaluru, N. Total synthesis of a functional designer eukaryotic chromosome. Science , 55—58 Ostrov, N. Design, synthesis, and testing toward a codon genome. Bugs 3 , — Hoess, R. The role of the loxP spacer region in P1 site-specific recombination. Nucleic Acids Res.
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Langmead, B. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Eyre-Walker, A. The distribution of fitness effects of new mutations. Attfield, P. Gibson, B. Yeast responses to stresses associated with industrial brewery handling. FEMS Microbiol. Bai, F. Ethanol fermentation technologies from sugar and starch feedstocks. Genome-wide identification of genes required for growth of Saccharomyces cerevisiae under ethanol stress. Yeast 23 , — Anderson, M. FEMS Yeast.
Alper, H. Engineering yeast transcription machinery for improved ethanol tolerance and production. Snoek, T. Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance. Voordeckers, K.
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Yoshikawa, K. Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae. Shi, D. Genome shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae. Caspeta, L. Altered sterol composition renders yeast thermotolerant. Science , 75—78 Graves, T.
Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash. Si, T. RNAi-assisted genome evolution in Saccharomyces cerevisiae for complex phenotype engineering. ACS Synth. Mumberg, D. Regulatable promoters of Saccharomyces cerevisiae : comparison of transcriptional activity and their use for heterologous expression. Ellis, T. Diversity-based, model-guided construction of synthetic gene networks with predicted functions.
Murphy, K. Combinatorial promoter design for engineering noisy gene expression. Duan, Z. A three-dimensional model of the yeast genome. Janke, C. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21 , — Kushnirov, V. Rapid and reliable protein extraction from yeast. Yeast 16 , — Download references. We thank Dr Erika Szymanski for proof-reading the manuscript. Correspondence to Yizhi Cai or Junbiao Dai.
Reprints and Permissions. Microbial Cell Factories Biotechnology for Biofuels Science China Life Sciences By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Article metrics. Advanced search. Skip to main content. Subjects Genome evolution Synthetic biology. Abstract SCRaMbLE is a novel system implemented in the synthetic yeast genome, enabling massive chromosome rearrangements to produce strains with a large genotypic diversity upon induction.
Introduction In the past decade, substantial progress has been made in the field of genome biology.
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Full size image. Stress tolerance test of the original synXII strain To identify a suitable stress condition that could significantly limit the growth of synXII strain but not so harsh to kill the cells, series dilution experiments were conducted. Phenotyping of the selected strains The selected strains were tested by either serial dilution as describe above or measuring the growth curve in the selected medium.
Data availability All data used for this paper are available from the authors on request. Additional information Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Article PubMed Google Scholar 7.
Article PubMed Google Scholar Electronic supplementary material Supplementary Information. Peer Review File. Description of Additional Supplementary Files. Supplementary Data 1. Supplementary Data 2. Many genes are known to influence cell fate specification in the Arabidopsis root epidermis. WER is expressed preferentially in the developing N-position cells and directly induces GL2 expression to specify the non-hair cell fate, whereas CPC inhibits GL2 expression in the H-position cells to specify the hair cell fate. Thus, the differentiating epidermal cells communicate with each other to achieve their proper cell-type pattern via the action of a mobile transcription factor CPC produced by WER-expressing N-cells to prevent neighboring H-cells from adopting the non-hair cell fate.
The intercellular movement of CPC is likely to occur through plasmodesmata PD , which provide cytoplasmic connections between plant cells 14 , In addition to intercellular trafficking, plant cells communicate with other cells and their environment through receptor-like kinases RLKs on the cell surface. In the developing root epidermis, SCM accumulates preferentially in the H-position cells through a feedback mechanism 20 , and has been proposed to respond to a positional signal and preferentially inhibit WER expression in the H-position cells However, it is not yet known how the initial difference in SCM activity between the N-position cell and the H-position cell is initiated.
Furthermore, it is not clear how SCM action leads to inhibition of WER expression in the H-position cell, considering that SCM kinase activity is not required for epidermal cell patterning 17 , To understand how SCM functions in root epidermal cell patterning, we used a genetic approach to search for new regulators acting in the SCM signaling pathway.
QKY, a multiple C2 domain and transmembrane region protein MCTP , has previously been reported to interact with SCM and affect epidermal cell patterning 22 , 23 , 24 , although its role in root epidermal cell patterning is unknown. We confirmed that this phenotype is caused by a single nuclear recessive mutation by analyzing the F1 and F2 offspring from a cross with wild-type plants. Through a bulk segregant analysis, we found that the mutation is linked to a marker nga on chromosome 1, which is near the QKY gene previously reported to affect root epidermal cell patterning Allelism testing by crossing this new mutant with qky-8 22 and with scm-2 16 showed that the mutant phenotype was complemented by scm-2 but not complemented by qky-8 Supplementary Fig.
Together, these results indicate that this newly identified mutant is a new allele of the QKY gene, and we named it qky In the qky mutant, the position-dependent expression pattern of these three genes was disrupted, causing reporter gene-expressing cells and reporter gene-non-expressing cells to be produced at each position Supplementary Fig. These results suggest that, like SCM, QKY acts early during root epidermis development to enable cells to interpret their relative position and appropriately regulate expression of the downstream transcription factor network.
These plants exhibit strong GUS accumulation in the stele cells and the ground tissue in the root meristematic region, as well as a lower GUS level in developing root epidermal cells and root cap cells Fig. Expression pattern and tissue-autonomous function of QKY gene in the root. The photos for each view were taken at a similar location in the root, respectively. Asterisks indicate the H-position cells. Therefore, we tested several versions of reporter constructs containing GFP in-frame with the QKY coding sequence, and found that a translational reporter construct with the GFP sequence inserted between the end of the first transmembrane domain and the beginning of the phosphoribosyltransferase PRT domain QKYp : QKY - GFP was able to completely rescue the qky mutant phenotype Supplementary Fig.
Due to the broad expression of QKY in root tissues and a report suggesting non-cell autonomous action of QKY in aerial tissues 23 , we tested the long-range action of QKY in regulating root epidermal cell patterning.
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We observed the fluorescence signal only in the tissue s where each promoter was expected to be active, and we found that only the WERp:QKY-GFP construct was able to rescue the qky mutant phenotype in the root epidermis Fig. These results indicate that QKY influences root epidermal cell patterning in a tissue autonomous manner. Notably, SCM has been reported to act in a cell autonomous manner in the root epidermis Given the similar mutant phenotypes, gene expression patterns, protein accumulation, and epidermis action for QKY and SCM , we sought to examine the relationship between these two genes.
First, we tested for possible genetic interaction by generating the qky scm-2 double mutant by a genetic cross. The abnormal epidermal cell pattern in this double mutant was not significantly different from the abnormal pattern in each of the single mutants Fig. Similarly, the double mutant exhibited disruption in the position-dependent expression of the GL2p : GUS marker that was comparable to the single mutants Fig. The disrupted cell patterning in the qky and scm mutants and the presumed localization of the QKY and SCM proteins at PD 23 led us to examine their possible role in the lateral inhibition between epidermal cells mediated by the mobile transcription factor CPC It was also shown that the scm-2 cpc-1 root phenotype was quite different from the scm-2 mutant phenotype The H-position epidermal cells are marked with an asterisk.
Quantitative analysis was performed using only sink cells in rosette leaves. Values indicate the percentage of cells showing GFP movement to the neighboring cells white and cells not showing GFP movement black. At least 76 cells were counted for each experiment. Actual number of cells analyzed in each experiment can be found in Supplementary Fig. Statistical significance of differences in the frequency was tested by chi-square test, and the P values for a, b, c, and d are 0. The construct combinations were cointroduced into the yeast strain AH Transformants were grown on the -L-T lacking leucine and tryptophan control plates and the -A-H-L-T lacking adenosine, histidine, leucine and tryptophan, selective plates for 3 days.
Mutations are shown in the right panel of a. Total protein extracts were subjected to immunoblotting using monoclonal anti-GFP antibodies. Their expression was confirmed by western blot analysis with total protein extracts using polyclonal anti-GFP antibodies. However, it was reported that the cytoplasmic domain of SCM interacts with the QKY D1, leading to the suggestion that this is a cytoplasmic domain We coexpressed these in Nicotiana benthamiana leaf epidermal cells and were able to detect EYFP fluorescence.
Together, these results suggest that the domain 1 and domain 3 of QKY are extracellular domains, and that domain 1 is used by QKY to interact with SCM and regulate epidermal cell patterning. Therefore, we examined the effect of MG an inhibitor of proteasome and cysteine protease , lactacystin a proteasome-specific inhibitor , and Ed a cysteine protease inhibitor on SCM-GFP protein level. Because Ed is known to inhibit the fusion of late endosomes with vacuoles in Arabidopsis 32 , we examined whether SCM-GFP accumulation in late endosomes increased after Ed treatment.
We also found that treatment of concanamycin A ConA a vacuolar ATPase inhibitor, which prevents the vacuolar degradation of proteins greatly increased the accumulation of SCM-GFP in the vacuole in the qky scm-2 mutant root, while it caused slightly increased vacuolar accumulation of SCM-GFP in the epidermis of the scm-2 mutant root Fig. Confocal images of the root epidermal cells left , and the quantified fluorescence intensities in the PM and in the inner area right are shown. P values for a, b, and c are 0.
Typical examples of images and scatterplots are shown left. Statistical significance was determined by unpaired t test, and P values for a is 0.
The level was also analyzed by western blot right. In each seedling, ten cells were examined. These results indicate that SCM is internalized from the PM by endocytosis and degraded in the vacuole, and that QKY prevents this internalization and vacuolar degradation. Same confocal detection setting was used to compare the fluorescence intensities from different plants. The protein level was also analyzed by western blot right.
Fluorescence intensities were measured in two adjacent cell files from the starting point of elongation zones left and the intensities of the four peaks in the N-position cells and the nearest four peaks in the H-position cells were compared right.
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Error bars indicate standard deviation from three independent transgenic lines. Five plants were analyzed in each transgenic line. Altogether, these results suggest that monoubiquitination, which is prevented by QKY in the H-position cells, is necessary for differential accumulation of SCM between cells at each position in the root epidermis. To identify the ubiquitination sites in the SCM protein, we analyzed the gel mobility of mutant versions of SCM-GFP with single or multiple lysine residues replaced by arginine residues and expressed transiently in qky mutant leaves Supplementary Fig.
These results suggest that SCM is modified by multi-monoubiquitination and that these three residues Lys, and at least one from Lys and Lys represent the ubiquitination site. However, the mechanism and regulation of SCM action had been unclear prior to this study. Like scm mutants, the qky mutant lacked position-dependent cell-type specification Supplementary Fig. PD are intercellular channels conferring cytoplasmic continuity between plant cells and controlling movement of various molecules by targeted and nontargeted mechanisms.
FTIP was shown to directly interact with FT in companion cells through its third C2 domain and to control FT export from companion cells to the neighboring sieve elements Accordingly, an important future goal is to explore the mechanism responsible for preferential H-position cell accumulation of QKY. In mammals and yeast, monoubiquitination and Kpolyubquination generally govern trafficking of membrane proteins, whereas Kpolyubiquitination generally regulates proteasome-dependent protein degradation 43 , In plants, diverse forms of ubiquitination appear to be used for the degradation of different membrane proteins.
Other membrane proteins have also been reported to be ubiquitinated. BOR1, a boron transporter protein, is mono- or diubiquitinated and degraded in the vacuole We found that SCM is multi-monoubiquitinated, and this promotes its internalization and vacuolar degradation Figs. Taken together, monoubiquitination seems to be sufficient for the internalization of membrane proteins in plants, as observed in nonplant organisms 43 , 44 , but multi-monoubiquitination and Kpolyubiquitination may increase the efficiency of internalization, as also shown in yeast 49 , 50 , and may be responsible for the vacuolar targeting.
It is possible that SCM is also polyubiquitinated, which may have gone undetected due to its low level. Here, however, we showed that blocking SCM degradation in vacuoles resulted in greater accumulation of multi-monoubiquitinated SCM, implying that multi-monoubiquitination plays a major role in the vacuolar degradation of SCM even though we cannot rule out the possibility of polyubiquitination.
Together, these results provide an updated model for cell fate specification in the Arabidopsis root epidermis. In this model, QKY protein preferentially accumulates in the H-position cells of the root epidermis and stabilizes SCM protein in these cells by preventing its ubiquitination. In turn, feedback regulation negative regulation by WER and positive regulation by CPC on SCM expression 20 further establishes preferential accumulation of SCM protein in H-position cells, which ultimately results in the cell-type pattern. The wer-1 , cpc-1 , scm-2 , and qky-8 alleles were described previously 7 , 8 , 16 , For plant growth, seeds were sterilized, germinated and grown vertically on agarose-solidified medium containing mineral nutrients For each line, we analyzed at least ten 4-day-old seedlings and performed three independent experimental repeats.
We scored the ten H-position cells and the ten N-position cells for each seedling using differential interference contrast microscopy.